Compositions and methods for enterohemorrhagic escherichia coli (ehec)vaccination

ABSTRACT

Certain embodiments are directed to compositions comprising EHEC-specific antigens. In certain aspects EHEC O157:H7-specific antigen(s) are used as components of immunogenic compositions and vaccines.

PRIORITY

This application claims priority to U.S. Provisional Patent Application Ser. No. 61/948,001 filed Mar. 4, 2014, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under R21AI09956001 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains are major human food-borne pathogens, responsible for bloody diarrhea and hemolytic uremic syndrome (HUS) worldwide. So far, there is no vaccine for humans against EHEC infections.

Enterohemorrhagic Escherichia coli (EHEC) strains are zoonotic extracellular pathogens, members of the Shiga-toxin producing E. coli (STEC) pathogroup. EHEC causes sporadic outbreaks of diarrhea and hemorrhagic colitis, particularly in developed countries [reviewed in (1-3)]. In the United States, EHEC causes approximately 0.9 cases per 100,000 inhabitants, with a significant number of hospitalizations and death, particularly among children and the elderly [reviewed in (4, 5)]. E. coli 0157:H7 comprises the serotype most commonly associated with outbreaks (6) and the expression of Shiga toxin (Stx), in addition to be linked to hemorrhagic colitis, it is associated with the progression to the hemolytic uremic syndrome (HUS), which cause renal failure and high fatality rate [reviewed in (7)]. In addition, EHEC 0157:H7 uses a type 3 secretion system (T3SS) to translocate effector proteins into the eukaryotic cell, causing changes in the host cytoskeleton, ultimately leading to improved bacterial adherence and colonization and, in some cases, host cell death (8). The EHEC T3SS is comprised of a basal A TP-dependent secretion apparatus, with an EscC polymer ring spanning bacterial outer membrane and a needle like structure formed by polymers of the EscF protein and an extension structure comprised of polymerized EspA. Finally, the EspD and EspB proteins form a translocon structure in the host membrane (9-11).

Generally asymptomatic, ruminants are the principal EHEC reservoir. Contaminated meat or fresh produce resulting from animal shedding constitutes an important route for human infection (12). Current prevention efforts are centered in the elimination of animal colonization, whether by vaccination or by improving sanitary and breeding practices (12, 13). Once the human infection is acquired, supportive care is provided, since antibiotic treatment could induce Shiga toxin expression. To date, two vaccines able to reduce EHEC colonization in cattle are commercially available (13, 14). Nevertheless, development of other subunit-based vaccines has been focused in the T3SS and its associated proteins, as well as Stx (4, 12). For example, inactivated Stx-derivatives are able to induce Stx-neutralizing antibodies in mice (15, 16) and hybrid A-B subunit-derived Stx toxins also induce antibody production and increase survival against toxemia and EHEC challenge in vivo (17-19). Fusion proteins comprising of Stx-derived peptides and T3SS-related proteins are promising vaccine candidates. St2B-Tir-Stx1 B-Zot, Stx2B-Stx1 B-lnt281, EspA-Stx2A 1, EspA-IntiminC300-Stx2B and Stx2B-BLS fusions have been demonstrated to reduce EHEC colonization in animal models, such as mice and goats (20-27). Overall, cumulative information indicates that mucosal delivery routes seem to be an effective way to induce immune responses to block the adhesion of EHEC in the intestine, mainly through expression of secretory lgA (slgA) (4).

In addition to the worldwide outbreaks caused by STEC 0157:H7, this organism has come recently under renewed scientific investigation as a result of the emergence of a subpopulation of strains that have acquired critical virulence factors that contribute to more severe and lethal disease in humans (28, 29). Further, the discovery of cattle reservoirs shedding high levels of STEC 0157:H7, which has been associated with the transmission between animals and across the human-animal interface (30, 31), strongly supports the idea that adoption of vaccination for livestock and/or susceptible individuals will have significant public health benefits, preventing substantial numbers of human STEC 0157 cases (32). Therefore, further discovery for EHEC-specific antigens needs to be done to improve existing or to develop novel vaccines.

SUMMARY

Because the EHEC-associated disease is complex and many molecular and cellular processes affected during infection are not fully understood, it is plausible to propose that some EHEC-encoded virulence-associated proteins could have important, yet unveiled role in the immune/protective process. Therefore, in order to bypass the bias towards assaying a limited number of known virulence factors as components of a vaccine against 0157:H7, the inventor performed a genome-wide in silico search for proteins most likely to be effective as immunogenic/protective antigens. By comparative genomics, EHEC-specific antigens were identified with high probability to be exposed to the host during infection. Using an immunoinformatics approach, the candidates were further grouped into high, medium, and low priority groups based on their putative antigenicity and screened as vaccine candidates in a murine model of gastrointestinal infection. Three candidates from each group were selected and evaluated as DNA vaccines for their capacity to induce an EHEC immune response and to reduce bacterial colonization in the murine intestine.

Certain embodiments are directed to compositions comprising EHEC-specific antigens. In certain aspects EHEC O157:H7-specific antigen(s) are used as components of immunogenic compositions and vaccines. Vaccines described herein are able to protect against pathogenic E. coli infections without affecting commensal flora. The inventor has identified Shiga toxin-producing E. coli (STEC) O157 specific antigens using comparative genomics. These STEC O157 specific antigens were identified as having a high probability of being exposed to the host during infection. Subsequently, an immunoinformatics approach was used to group STEC O157 specific antigens into priority groups (high, medium and low) based on their antigenicity. Screening of these identified antigens in a murine model of gastrointestinal infection demonstrated STEC-specific immune responses and a reduction in STEC colonization of the murine intestine particularly associated with the high priority group. These high priority antigens displayed protective properties or vaccination properties as tested in the murine model of infection.

In certain aspects the antigens described herein can be formulated in an immunogenic or vaccine composition. The composition may include an adjuvant and/or an anti-microbial agent.

Certain embodiments are directed to a composition that stimulates an immune response against EHEC antigens. In certain aspects the immune response can be a therapeutic immune response treating an infection in a mammal or a protective immune response preventing EHEC related disease in a mammal. In certain aspects the compositions provide a vaccine effective to reduce, prevent and/or eliminate EHEC colonization of a ruminant or other mammal.

In certain aspects the induced immune response reduces shedding EHEC into the environment from a treated mammal. In certain aspects the mammal is an infected mammal, such as a bovine, an ovine, a suinae, or homo sapien (human). In a further aspect the administration of the antigen compositions can reduce EHEC contamination of the environment, food products, and/or water.

Certain embodiments are directed to a vaccine composition comprising an enterohemorragic E. coli (EHEC) specific antigen(s) and an immunological adjuvant. In certain embodiments, the EHEC is EHEC O157:H7, EHEC EDL933, EHEC Sakai, and/or STEC O157. In additional embodiments, the immunological adjuvant comprises an oil-in-water emulsion, such as a mineral oil and dimethyldioctadecylammonium bromide. In yet additional embodiments, an adjuvant is present at a concentration of about 20% to about 40% (v/v), such as at a concentration of 30% (v/v).

Further embodiments are directed to methods for eliciting an immunological response in a mammal against an enterohemorragic E. coli (EHEC) antigen. In certain aspects the method comprises administering to a mammal a therapeutically effective amount of a composition comprising a nucleic acid encoding an EHEC antigen that is expressed when introduced to a mammal. In additional embodiments, the mammal is a human or a ruminant, such as a bovine. In yet further embodiments, the composition further comprise an adjuvant.

Still further embodiments are directed to methods for reducing colonization of enterohemorragic E. coli (EHEC) in a mammal comprising administering to the mammal a therapeutically effective amount of a composition comprising an EHEC antigen as described herein.

As used herein, the term “antigen” is a molecule capable of being bound by an antibody or T-cell receptor. An antigen is additionally capable of inducing a humoral immune response and/or cellular immune response leading to the production of B- and/or T-lymphocytes. The structural aspect of an antigen, e.g., three-dimensional conformation or modification (e.g., phosphorylation), giving rise to a biological response is referred to herein as an “antigenic determinant” or “epitope.” B-lymphocytes respond to foreign antigenic determinants via antibody production, whereas T-lymphocytes are the mediator of cellular immunity. Thus, antigenic determinants or epitopes are those parts of an antigen that are recognized by antibodies, or in the context of an MHC, by T-cell receptors. An antigenic determinant need not be a contiguous sequence or segment of protein and may include various sequences that are not immediately adjacent to one another. In certain embodiments, binding moieties other than antibodies and be engineered to specifically bind to an antigen, e.g., aptamers, avimers, and the like.

The term “antibody” or “immunoglobulin” is used to include intact antibodies and binding fragments/segments thereof. Typically, fragments compete with the intact antibody from which they were derived for specific binding to an antigen. Fragments include separate heavy chains, light chains, Fab, Fab′ F(ab′)2, Fabc, and Fv. Fragments/segments are produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins. The term “antibody” also includes one or more immunoglobulin chains that are chemically conjugated to, or expressed as, fusion proteins with other proteins. The term “antibody” also includes bispecific antibodies. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai and Lachmann, Clin Exp Immunol 79:315-21, 1990; Kostelny et al., J. Immunol. 148:1547-53, 1992.

The term “isolated” can refer to a nucleic acid or polypeptide that is substantially free of cellular material, bacterial material, viral material, or culture medium (when produced by recombinant DNA techniques) of their source of origin, or chemical precursors or other chemicals (when chemically synthesized). Moreover, an isolated compound refers to one that can be administered to a subject as an isolated compound; in other words, the compound may not simply be considered “isolated” if it is adhered to a column or embedded in an agarose gel. Moreover, an “isolated nucleic acid fragment” or “isolated peptide” is a nucleic acid or protein fragment that is not naturally occurring as a fragment and/or is not typically in the functional state.

As used herein, the term EHEC “antigen” refers to a polypeptide identified in Table 1.

As used herein, a “recombinant” EHEC secreted protein refers to the full-length polypeptide sequence, fragments of the reference sequence or substitutions, deletions and/or additions to the reference sequence, so long as the proteins retain effectiveness in the animal models described herein. Generally, variants of an antigen will display at least about 90% sequence identity, preferably at least about 95% to 99% sequence identity to the full-length reference sequence or an immunogenic portion thereof. Percent sequence identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M. O. in Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5 Suppl. 3:353-358, National biomedical Research Foundation, Washington, D.C., which adapts the local homology algorithm of Smith and Waterman (1981) Advances in Appl. Math. 2:482-489 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.

As used herein, “colonization” refers to the presence of EHEC in the intestinal tract of a mammal.

As used herein, “shedding” refers to the presence of EHEC in feces.

As used herein, “therapeutic amount”, “effective amount” and “amount effective to” refer to an amount of vaccine effective to elicit an immune response against a secreted antigen, thereby reducing or preventing EHEC disease, and/or EHEC colonization of a mammal, e.g., a ruminant or human; and/or reducing the shedding of EHEC; and/or reducing the number of EHEC shed by a mammal; and/or, reducing the time period of EHEC shedding by a mammal.

As used herein, “immunization” or “immunize” refers to administration of an immunogenic composition in an amount effective to stimulate the immune system of an animal or mammal to which the composition is administered and elicit an immunological response against one or more antigens present in the composition.

An “immunological response” to a composition or vaccine is the development in the host of a cellular and/or antibody-mediated immune response to the composition or vaccine of interest. Usually, an “immunological response” includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells and/or γδ T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective immunological response such that EHEC disease is lessened and/or prevented; resistance of the intestine to colonization with EHEC is imparted; shedding of EHEC is reduced; the number of EHEC shed by a mammal is reduced; and/or the time period of EHEC shedding by a mammal is reduced.

The terms “immunogenic” protein or polypeptide refer to an amino acid sequence that elicits an immunological response as described above. An “immunogenic” protein or polypeptide, as used herein, includes the full-length sequence of the particular EHEC protein in question, analogs thereof, aggregates, or immunogenic fragments thereof. By “immunogenic fragment” is meant a fragment of a secreted EHEC protein that includes one or more epitopes and thus elicits the immunological response described above. Such fragments can be identified using any number of epitope mapping techniques well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715, all incorporated herein by reference in their entireties. Similarly, conformational epitopes are identified by determining spatial conformation of amino acids (e.g., using x-ray crystallography and 2-dimensional nuclear magnetic resonance). See, e.g., Epitope Mapping Protocols, supra. Antigenic regions of proteins can also be identified using standard antigenicity and hydropathy plots, such as those calculated using, e.g., the Omiga version 1.0 software program available from the Oxford Molecular Group. This computer program employs the Hopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci USA (1981) 78:3824-3828 for determining antigenicity profiles, and the Kyte-Doolittle technique, Kyte et al., J. Mol. Biol. (1982) 157:105-132 for hydropathy plots.

“Recombinant” polypeptides refer to polypeptides produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide. “Synthetic” polypeptides are those prepared by chemical synthesis.

The term “treatment” as used herein refers to either (i) the prevention of infection or reinfection (prophylaxis), or (ii) the reduction or elimination of symptoms of the disease of interest (therapy).

By “mammalian subject” is meant any member of the class Mammalia, including humans and other mammary gland possessing animals, such as ruminants, including, but not limited to, bovine, porcine, and Ovis (sheep and goats) species.

Moieties of the invention, such as polypeptides, peptides, antigens, or immunogens, may be conjugated or linked covalently or noncovalently to other moieties such as adjuvants, proteins, peptides, supports, fluorescence moieties, or labels. The term “conjugate” or “immunoconjugate” is broadly used to define the operative association of one moiety with another agent and is not intended to refer solely to any type of operative association, and is particularly not limited to chemical “conjugation.”

The term “providing” is used according to its ordinary meaning “to supply or furnish for use.” In some embodiments, the protein is provided directly by administering the protein, while in other embodiments, the protein is effectively provided by administering a nucleic acid that encodes the protein. In certain aspects the invention contemplates compositions comprising various combinations of nucleic acid, antigens, peptides, and/or epitopes.

The phrase “specifically binds” or “specifically immunoreactive” to a target refers to a binding reaction that is determinative of the presence of the molecule in the presence of a heterogeneous population of other biologics. Thus, under designated immunoassay conditions, a specified molecule binds preferentially to a particular target and does not bind in a significant amount to other biologics present in the sample. Specific binding of an antibody to a target under such conditions requires the antibody be selected for its specificity to the target. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.

The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

FIG. 1. Schematic summary representation of the comparative genomics and immunoinformatics approaches used in the studies described herein for the identification of vaccine candidates from EHEC O157:H7.

FIG. 2A-2F. EHEC-specific immune responses after vaccination and bacterial counts of EHEC infected mice. (A) EHEC-specific serum IgG and (B) fecal IgA following intranasal immunization. High priority (HP), Medium priority (MP) and Low priority (LP) vaccines with Cholera toxin (CT) as adjuvant were used. Controls included TE, pVAX-1 or CT. (C) Cecum segments were obtained from HP, MP and LP vaccinated mice 3 days after infection with EHEC O157:H7. The bar graph depicts log CFU/g of cecum. (D) EHEC-specific IgG antibody titers detected in serum and (E) IgA in feces from mice immunized with individual HP priority group candidates pVAX-12, pVAX-31, and pVAX-56.2. (F) Cecum segments were obtained from each vaccinated mice and bacterial counts determine 6 days post-infection. Asterisks represent statistical significant differences.

FIG. 3A-3B. Schematic heat map representation of cytokine, chemokines and growth factors in response to vaccinated and/or infected mice. (A) Bio-plex assays were done in sera of immunized mice administered with HP, MP, and LP priority candidates and (B) individual vaccine candidates: pVAX-12, pVAX-31 and pVAX-56.2. Non-immunized sera from TE and CT mice were included as controls. Data are Z-score transformed and relative to controls and expressed as standard deviation (SD) units from the row mean. The samples were diluted up to 1:1 for the assay. Results are expressed as Mean±SD of duplicates. Scale for SD deviation is shown at the bottom of the heat map. Statistically significance was calculated in both the A and B groups using one-way ANOVA. All the cytokines presented in the heat map were significantly different (p<0.001) compared to TE group.

DESCRIPTION

Most EHEC subunit vaccine candidates tested to date are comprised of known virulence factors, such as Stx and the T3SS-related proteins. These virulence factors are well characterized and known to be essential for the onset of EHEC colonization and/or host damage. Further, it is well documented that the main protection mechanism for these vaccine candidates is the induction of neutralizing antibodies (4). However, the DNA sequences encoded in the genome of EHEC strains may contain unveiled genes encoding antigenic proteins that have not yet been investigated as vaccine candidates. Computational vaccinology tools have been proposed as a potentially powerful aid in vaccine development, particularly for new or emerging pathogens from which critical antigenic determinants and/or virulence factors knowledge is limited (56). The methods described herein combines comparative genomics and immunoinformatics analysis of available EHEC genomes in the search for vaccine candidates. This approach represents an unbiased screening method, as it seeks in the encoded sequences irrespective of their putative or experimental function, which allows the discovery of potential candidates overlooked by other EHEC vaccine studies. Subsequently, this method led to a list of 65 vaccine candidates, three of which were proven to be able to induce immune responses, and one of them also reduced EHEC colonization when delivered as a DNA vaccine.

Enterohemorrhagic E. coli (EHEC) O157:H7 strains are major human food-borne pathogens, responsible for bloody diarrhea and hemolytic uremic syndrome (HUS) worldwide. So far, there is no vaccine for humans against EHEC infections. In the studies described herein, a comparative genomics analysis was performed to identify EHEC-specific antigens useful as potential vaccines. The genes are present in both EHEC EDL933 and Sakai strains but absent in non-pathogenic E. coli K-12 and HS strains. The EHEC genes were subjected to an in silico analysis to identify secreted or surface-expressed proteins. The inventor identified 65 gene-encoding protein candidates that were subjected to immunoinformatics analysis. The candidates were categorized as high priority (HP), medium priority (MP), and low priority (LP). Three members of each group were selected and cloned into pVAX-1. Candidates were pooled accordingly to their priority group and tested for immunogenicity against EHEC O157:H7 using a murine model of gastrointestinal infection. The high priority (HP) pool, containing genes encoding for a Lom-like protein (pVAX-31)(SEQ ID NO:1), a putative pilin subunit (pVAX-12)(SEQ ID NO:2), and a fragment of the type III secretion structural protein EscC (pVAX-56.2)(SEQ ID NO:3), was able to induce the production of EHEC specific lgG and sIgA in sera and feces. HP-immunized mice displayed elevated levels of Th2 cytokines and diminished cecum colonization after wt challenge. Individually tested HP-vaccine candidates showed that pVAX-12 and pVAX-56.2 significantly induced Th2 cytokines and production of fecal EHEC sIgA, with pVAX-56.2 reducing EHEC cecum colonization. The bioinformatics approach described herein is able to identify vaccine candidates useful to prevent EHEC O157:H7 infections.

As intestinal mucosal surfaces are composed of exposed tissue in permanent contact with harmless environmental bacteria, which likely participate in maintenance of a homeostasis state (49), immune responses are relatively difficult to induce by vaccine candidates delivered in the gastrointestinal mucosa. The HP antigens described herein were able to induce EHEC humoral responses both in serum and in intestinal mucosa. Although some candidates assayed in mice have been shown to be protective by inducing exclusively serum responses (20, 57), most involved the production of sIgA (17, 58-61). In this case, the induction of sIgA by the HP pool correlated with a reduction in colonization, while the MP pool was also able to increase serum IgG levels but failed to reduce EHEC colonization after challenge. Assays performed with the three representative HP candidates individually also seem to support the role of intestinal mucosa humoral response in protection, as pVAX-12 failed to protect against EHEC colonization, despite inducing a strong serum humoral response. On the other hand, pVAX-56.2, the candidate inducing the highest sIgA titers in feces, was able to cause a reduction of bacterial load in cecum. In certain aspects the combined immune response induced by the three representative HP antigens achieves colonization reduction levels.

Certain embodiments will employ conventional techniques of molecular biology, microbiology, recombinant DNA technology, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Vols. I, II and III, Second Edition (1989); Perbal, B., A Practical Guide to Molecular Cloning (1984); the series, Methods In Enzymology (S. Colowick and N. Kaplan eds, Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds, 1986, Blackwell Scientific Publications).

I. Methods for Antigen Identification

Subunit EHEC vaccine research has been mainly focused on known virulence factors. The inventor sought to screen the genome of EHEC strains EDL933 and Sakai to identify sequences with a high probability to encode protective antigens, independently of their previous assigned function. Mucosal immune responses are relatively difficult to induce by vaccine candidates due to the fact that mucosal surfaces are often in contact with non-pathogenic E. coli, which enhance the homeostatic state [reviewed in (49)]. Thus, a first criterion was to identify common EHEC antigens absent in non-pathogenic E. coli strains. For this, the set of common proteins between EDL933 and Sakai 0157:H7 EHEC strains were determined by a reciprocal BLAST analysis as described below. Next, those DNA sequences encoding proteins present in the non-pathogenic E. coli K-12 MG1655 and commensal HS strains genomes were eliminated from the pool. This analysis rendered 897 protein sequences. It is proposed that the probabilities to provide protection increase for those antigens detected during natural infection, meaning that bacterial-exposed proteins comprise better vaccine candidates (50). In order to predict their subcellular localization, the 897 proteins selected in the previous analysis were fed to the P-SORTb software (available on the worldwide web at psort.org/psortb/). A total of 65 proteins putatively associated with the outer membrane and/or secreted were selected as vaccine candidates (Table 1).

TABLE 1 EHEC O157-specific secreted and outer membrane associated proteins identified by P-SORT analysis. # GI number Annotation 1 12512859 putative fimbrial protein 2 12512975 hypothetical protein Z0266 3 12513130 putative beta-barrel outer membrane protein 4 12513211 putative structural protein (partial) 5 12513363 putative outer membrane export protein 6 12513364 hypothetical protein Z0609 7 12513368 putative RTX family exoprotein 8 12513376 hypothetical protein Z0639 9 12513752 putative outer membrane protein of prophage CP-933K 10 12514345 putative outer membrane protein Lom precursor of protein bacteriophage BP-933W 11 12514376 hypothetical protein Z1516 12 12514403 putative pilin subunit 13 12514410 putative member of ShiA/HecA/FhaA exoprotein family 14 12514411 putative outer membrane transporter of ShiA/HecA/FhaA exoprotein family 15 12514503 Putative receptor 16 12514836 putative tail component of prophage CP-933X 17 12514898 putative outer membrane receptor, probably tonB dependent 18 12515102 putative tail component of prophage CP-9330 19 12515159 orf, hypothetical protein 20 12515160 putative ATP-bindingcomponent of a transportsystem and adhesion protein 21 12515311 putative outer membrane protein 22 12515315 hypothetical protein Z2323 23 12515551 putative chaperone protein 24 13259573 putative Lom-like outer membrane protein of cryptic prophage CP-933P 25 13259574 putative tail component of cryptic prophage CP-933P 26 13259580 putative tail component of cryptic prophage CP-933P 27 12516024 flagellar biosynthesis; flagellin, filament structural protein 28 12516037 putative secreted protein 29 12516039 putative secreted protein 30 12516089 unknown protein encoded within prophage CP-933U 31 12516092 putative outer membrane protein of prophage CP-933U 32 12516149 putative integrase for prophage CP-933U 33 12516174 putative outer membrane receptor for iron compound or colicin 34 12516360 putative Lom-like outer membrane protein of prophage CP-933V 35 12516361 putative tail fiber protein of prophage CP-933V 36 12516373 putative major tail subunit encoded within prophage CP- 933V 37 169822942 putative fimbria! usher 38 12517052 hypothetical protein Z3920 39 12517087 hypothetical protein Z3954 40 12517088 putative enzyme 41 12517355 putative lipoprotein of type I11 secretion apparatus 42 12517375 type I11 secretion apparatus protein 43 12517526 putative PagC-Iike membrane protein 44 12517607 putative iron compound receptor 45 12518206 outer membrane heme/hemoglobin receptor 46 12518273 putative fimbria! subunit 47 12518274 putative fimbria! protein 48 12518278 putative major fimbria! subunit 49 12518349 putative adhesin 50 12518435 espF 51 12518439 secreted protein EspB 52 12518440 secreted protein ExpO 53 12518447 intimin adherence protein 54 12584449 putative translocated intiminreceptor protein 55 12518464 escJ 56 12518466 escC 57 12518483 hypothetical protein Z5142 58 12518576 putative fimbria! protein 59 12518577 putative fimbria! protein 60 12518578 putative fimbria! usher 61 12518581 putative major fimbria! subunit 62 12518689 hypothetical protein Z5335 63 3822134 putative exoprotein-precursor 64 3822145 hypothetical protein 65 3822162 hypothetical toxin protein Nomenclature: *low priority; **medium priority; ***high priority candidates. ^(a)We have further analyzed these final candidates using blastp and found paralogs of some of these sequences in recently sequenced non-pathogenic E. coli strains.

Further, the process of identifying highly antigenic vaccine candidates was accelerated by using immunoinformatics, which allowed the assignment of priorities for the testing of these vaccine candidates in the mouse model of EHEC O157:H7 infection. As described in FIG. 1 and based on physicochemical properties, the proteins were prioritized on the basis of either possessing no transmembrane (TM) domains or containing one region only, having a signal peptide or whether its localization was predicted to be as ‘secreted’, and whether they display a high score for adhesiveness. Proteins that satisfy more of these characteristics were ranked higher than others. The VaxiJen server (44) predicted the protective bacterial antigens based on the overall immunogenicity score (higher was better), and also helped to rank the proteins. For B-cell epitope predictions, three parameters were calculated from results: (i) total number of epitopes per sequence, (ii) total score of all epitopes combined per sequence, and (iii) average score of an epitope. Similarly, for T-cell epitope predictions, parameters such as (i) total number of high-binding (HB) epitopes, (ii) total score of HB epitopes, (iii) percentage of HB epitopes among all epitopes predicted for a given sequence, and (iv) number of human leukocyte antigens (HLA) alleles covered by the epitopes of a given sequence, were also calculated. These parameters were also taken into account when ranking the proteins; however, a larger weight was assigned to 8-cell epitopes since EHEC is an extracellular pathogen. The final ranking of all 65 proteins was conducted using a cumulative score from both physicochemical and immunological properties. Based on the combined informatics analysis, vaccine candidates were divided in three groups: High Priority (HP, 25 candidates), Medium Priority (MP, 28 candidates) and Low Priority (LP, 12 candidates) (indicated by asterisks in Table 1).

To screen the putative protective candidates as determined by their priority score, three representative candidates were randomly selected from each group (candidates 31, 56.2 and 12 from HP group; 43, 16 and 9 from MP group; and 51, 49.1 and 49.2 from LP group) and the selected representatives were cloned into pVAX1 DNA vaccine vector (51, 52). DNA vaccine construction comprises an approach that allows the rapid testing of several vaccine candidates instead of limited the analysis to antigens requiring optimized expression and purification. Being EHEC an extracellular intestinal pathogen, the approach sought to induce mucosal immune responses and DNA vaccines have been shown to induce both mucosal and systemic immune responses against pathogenic bacteria antigens when delivered by the intranasal route (53-55). During the cloning process in pVAX-1, large DNA sequences were divided in coding sequences with a maximum length of 1 kb. The constructs were pooled in their respective priority group and delivered intranasally in groups of 10 mice. A schedule of priming and two boosts was followed, with 60 μg of the pooled plasmids (20 μg of each individual construct) and cholera toxin (CT) as an adjuvant, which is further detailed in the methods described below. Next, induction of immune responses was evaluated in vaccinated mice. First, the production of EHEC IgG was monitored in post immunized (p.i.) mice by ELISA using serum samples collected one week after the last boost. The HP- and MP-immunized mice presented higher levels of EHEC IgG compared to animals receiving buffer (TE), adjuvant (CT) alone or pVAX-1 (empty vector) (FIG. 2). Next, EHEC IgA titers in feces were determined. The HP-vaccinated mice elicited higher titers of EHEC specific sIgA compared to MP- and LP-immunized animals and control groups (FIG. 2), though the titer declined with increased sera dilution. These results showed that although both HP and MP DNA vaccine pools induce EHEC antibody responses, only the HP group induced a higher amount of EHEC sIgA.

Two weeks after the last boost, animals were challenged with EHEC strain 86-24. Bacterial load in feces and cecum was determined from days 3-6 after infection. Results consistently showed a reduction pattern in the CFU recovered from feces in the HP vaccinated mice at days 4, 5 and 6 (FIG. 2) compared to TE buffer, CT, or pVAX-1 control groups. In addition, a reduction in the LP vaccinated group was observed at day 6, though at lower extent than that obtained in the HP group. Cecum organ platting revealed a significant reduction of over one log (P<0.05) in bacterial burden in HP vaccinated mice compared to the empty vector control group at day 3 (FIG. 2). No reduction was observed in the cecum of mice vaccinated with the MP- or LP-vaccinated pools. Overall, data showed that HP plasmids were more efficient in reducing EHEC colonization in the murine model.

To determine the extent of the immunogenic/protective effect of each candidate in the overall HP vaccine group, an individual plasmid vaccination experiment was performed. 60 μg of pVAX-12, pVAX-31, pVAX-56.2, or TE buffer were administered to groups of mice with the same vaccination schedule as the previous experiment. To assess the immunogenicity of individual DNA vaccines, mice sera were collected and assayed for EHEC IgG. Results showed increased EHEC IgG levels in pVAX-12. The IgG levels were also elevated with pVAX-56.2 vaccinated group compared to TE control group (FIG. 2). The pVAX-31 plasmid failed to induce the production of EHEC IgG. In parallel, EHEC sIgA was measured in feces of all vaccinated animals to evaluate mucosal immune induction. The mice immunized with pVAX-56.2 showed increases in sIgA up to the 1:64 dilution (FIG. 2), as compared to control animals. The pVAX-31 and, to a minor extent, pVAX-12 also increased the levels of sIgA in feces compared to TE control group. Overall, pVAX-12 produced the higher induction of serum IgG, while pVAX-56.2 was able to stimulate high titers of both serum IgG and sIgA.

Next, mice vaccinated with individual candidates were challenged with EHEC 86-24 two weeks after the last boost. Daily bacterial load in feces and cecum colonization from days 3 through 6 post infection were assessed. Results show a clear reduction of EHEC in feces in all vaccinated groups at day 6 (FIG. 2). No differences in cecum colonization are observed at day 3 (data not shown). However, a small reduction in EHEC CFU on the cecum from the pVAX-56.2 immunized group was detected at day 6 (FIG. 2). Overall, pVAX-56.2 consistently induced EHEC serum IgG and fecal sIgA and reduced EHEC shedding and colonization, suggesting that candidate 56.2, which encodes the C-terminal fragment of EscC was the best candidate tested.

Because the cellular immune response plays a leading role in protection and long-term immune response, the expression of cytokines in sera of HP-, MP- and LP-immunized and non-immunized mice were evaluated using a Bio-Plex assay. Eleven out of 23 inflammatory mediators were increased in HP-immunized mice compared to non-immunized mice, including the Th1 cytokines IL-1β, and TNF-α; the Th2 cytokines IL-3, IL-5, IL-6 and IL-10; the chemokines MIP-1α, MIP-1β, eotaxin, KC and MCP-1 and the growth factor GM-CSF (data summarized by heterogeneous color between columns in the heat map; FIG. 3A). Pro-inflammatory cytokines such as IL-1β, TNF-α and IL-6 are increased at an early stage of immune response and play a central role in the host defense mechanism. However, increased levels of Th2 cytokines, including IL-3, IL-5, IL-6, IL-10, but not IL-4 in HP immunized mice were also detected. It is proposed that these Th2-type cytokine responses maybe accounting for the generation of the humoral antibody response. Sera obtained from mice vaccinated with individual candidates pVAX-12 and pVAX-56.2 showed an increase of Th1 cytokines (IL-1β, IL-12p70, TNF-α), Th2 cytokines (IL-3, IL-5, IL-6 and IL-10), chemokines (MIP-1β, eotaxin, MCP-1, RANTES) and the GM-CSF growth factor compared to control mice (FIG. 3B).

Bacterial Strains and Media.

The prototype enterohemorrhagic E. coli O157:H7 strains 86-24 (33) and EDL933 (34) were grown in Luria Bertani (LB) broth at 37° C. Strains bearing pVAX1-derivatives were grown in medium supplemented with kanamycin (Sigma, 25 μg/ml) as requirement for recombinant plasmid selection.

Comparative Genomics and Immunoinformatics Analysis.

A comparative bioinformatic analysis was developed to obtain groups of orthologous proteins of E. coli O157:H7 strains that excluded orthologous proteins from other non-pathogenic (named external) strains. To achieve this, two E. coli O157:H7 strains (EDL933 and Sakai) were included and two external strains: E. coli HS and E. coli K-12 strain MG1655. The most probable set of orthologous proteins shared by the two E. coli O157:H7 strains were identified using a reciprocal best-hit criterion as follows. All the predicted proteins of one genome were searched against the predicted proteins of the other genome and vice versa using BLAST-P with a cutoff e-value of 10⁻¹². To be included in an ortholog group, the alignment region between the subject protein and the query protein had to be at least of 80%, and there had to be at least 40% similarity for both query and target sizes. Next, the orthologs groups that contained a protein of any of the two external strains were excluded (FIG. 1). This resulted in a set of 897 groups of orthologous proteins shared only by the E. coli O157:H7 strains. The ortholog sequences corresponding to the EDL933 strain were analyzed by the P-SORT software (available on the worldwide web at psort.org/psortb/) in order to predict their subcellular localization. Sixty five proteins that were putatively secreted or outer membrane-associated were selected as candidates. These proteins were extensively analyzed for critical features found in vaccine candidates such as physicochemical properties, adhesiveness and antigenicity, and subsequently predicted for immunodominant epitopes. At least two different software programs were used for each property as described below. In most cases, the underlying principle of these programs for the same categorical property is different to ensure wide coverage yet maintaining stringency by creating a consensus of predicted results (Table 2).

TABLE 2 Overview of prediction programs used. Physicochemical properties Immunological properties Transmembrane Signal- Sub-cellular Adhesion B-cell T-cell (TM)* peptide (SP) localization Probability* epitopes epitopes Antigenicity 1 TMHMM Signal-P Signal-P SPAAN ABCPred NetCTL VaxiJen (trained (trained (linear) (MHC-I) on Gram on Gram negative negative bacteria) bacteria) 2 HMMTOP PsortB PsortB LipoP NetMHCII (MHC-II) 3 PHOBIUS NetChop NetChop Lipo *Consensus of results from all programs in a given category, wherever applied.

The transmembrane (TM) regions were predicted using TMHMM (35), HMMTOP (36) and Phobius (37). Phobius can discern TM topology and signaling peptide in a protein. It substantially reduces the errors in the predictions of these two characteristics when compared to other algorithms including TMHMM. After an initial analysis with PsortB (38), the signal or localization peptides were also predicted using SignaiP (39) and NetChop (40). The presence of lipoprotein signal peptide was determined using the method described by Juncker et al (41), which predicts both Spl and Spll signal peptidases. Lipo program (42) recognizes the lipo-box in protein sequences, was also used. SPAAN (43) predicts the probability of a protein being an adhesin, which often comprise important factors in bacterial virulence. VaxiJen (44), an alignment-free approach for antigen prediction was used to score the overall antigenicity of the protein. VaxiJen model used was “bacteria” with threshold set to 0.5. 8-celllinear epitopes were predicted using ABCPred (45) while NetCTL (46) and NetMHC-II (47) programs were used to predict MHC class I and class II binding peptides, respectively. The ABCPred provided an accuracy of 65.93% and equal sensitivity and specificity using window length of 16-mer peptides with overlapping allowed. NetCTL predicts CTL epitopes restricted to 12 MHC class I supertypes and a specificity of 97% was used. MHC-II was used with an epitope length of 15 residues against 14 HLA-DR alleles covering the nine HLA-DR, six HLA-DQ and six HLA-DP supertypes. The overall strategy and its preferred outcome are outlined in FIG. 1.

Plasmid Construction.

Plasmid pVAX1™ was obtained from Invitrogen/Life Technologies (New York, USA). For this study, large candidates were subdivided in coding sequences (CDS) of maximum 1000 bp in length. For the high priority pool, candidates 31, 56, and 12 were selected. The candidate 56 (EscC) was divided in 2 CDS. As the amino terminal portion of EscC is oriented towards the periplasm (10), the fragment comprising the carboxy-terminus (pVAX56.2) was selected for testing. Candidates 43, 16, and 9 were selected for medium priority pool. Finally, candidate 51 and two CDS of the candidate 49 for the low priority pool were selected. The CDS for selected candidates were amplified by PCR from genomic DNA of E. coli EDL933 (O157:H7) using the corresponding forward (Fw) and reverse (Rv) primers containing HindIII and XhoI restriction sites, respectively (Table 2). The resulting fragments were digested and cloned into the HindIII and XhoI sites of pVAX 1. Fw primers were designed to generate a Kozak consensus sequence (ACCATGG) at the 5′-end of each CDS. All of the clones were sequenced.

TABLE 3 Primers used for plasmid construction. EHEC gene Construct insert Primers Sequence 5′-3′ pVAX-12 Z1538 PVAX12-Fw ACCAAGCTTACCATGGTTTCTACTTTCAAAAAAGC AG (SEQ ID NO:4) PVAX12-Rv ACCCTCGAGTAGAGGTAGCTCAGGGTGTATTCT (SEQ ID NO:5) pVAX-31 Z3075 PVAX31-Fw ATTAAGCTTACCATGGGTAAACTTTATGCCGCCAT TTTG (SEQ ID NO:6) PVAX31-Rv ATTCTCGAGTCAATGATGATGATGATGATGGAACT TATAACCGACACCCAC (SEQ ID NO:7) pVAX-56.2 a.a. 253- PVAX56.2-Fw ACCAAGCTTACCATGGACCGCGAAATAACGATGG 512 escC AT (SEQ ID NO:8) PVAX56.2Rv ACCCTCGAGTTATTCGCTAGATGCAGATTTTATC (SEQ ID NO:9) pVAX-43 Z4321 PVAX43-Fw ATTAAGCTTACCATGGGTGGTTCAAGACTGGCTGA TAATC (SEQ ID NO:10) PVAX43-Rv ATTCTCGAGTTAAAAACGATAACCAACTCCAAC (SEQ ID NO:11) pVAX-16 Z1908 PVAX16-Fw ATTAAGCTTACCATGGCTTTTTCTTTTTTTTCTACA AAACCCATACC (SEQ ID NO:12) PVAX16-Rv ATTCTCGAGTTATCCGCCCGCACCATTAACC (SEQ ID NO:13) pVAX-9 Z0981 PVAX9-Fw ACCAAGCTTACCATGGGTAAAGTTTGTGCAGCAA (SEQ ID NO:14) PVAX9-Rw ACCCTCGAGTCAAAATTTATAACCGACACCCAC (SEQ ID NO:15) pVAX-51 espB PVAX51-Fw ATTAAGCTTACCATGGATACTATTGATAATACTCA AG (SEQ ID NO:16) PVAX51-Rv ATTCTCGAGTCAATGATGATGATGATGATGCCCAG CTAAGCGACCCGATTG (SEQ ID NO:17) pVAX-49.1 a.a. 641- PVAX49.1-Fw ACCAAGCTTACCATGGCCGATGCCGTTAACGGCTC 960 ehaG (SEQ ID NO:18) PVAX49.2-Rv TTATCTAGACTCGAGTTACTCGGCGTTCGCAATGG TG (SEQ ID NO:19) pVAX-49.2 a.a. 1141- PVAX49.2-Fw ACCAAGCTTACCATGGAACTGCTCGGTGCATTGT 1380 ehaG CT (SEQ ID NO:20) PVAX49.2-Rv TTATCTAGACTCGAGTTAGCCGGAACCAATCGCG ACG (SEQ ID NO:21)

Immunization Protocol.

Six to eight week-old female BALB/c mice (Harlan Laboratories) were divided into six groups (n=10). Mice were immunized intranasally with 20 μg of the DNA vaccines in Tris-EDTA (TE) buffer (10 μl in each nostril), arranged as followed: (1) Tris-EDTA (TE) buffer only, (2) TE plus cholera toxin (CT, adjuvant), (3) pVAX vector plus CT, (4) pVAX-High Priority (HP), (5) pVAX-Medium Priority (MP), and (6) pVAX-Low Priority (LP) vaccine candidates. All the vaccine candidates were administered in pools of 3 targets (20 μg of each plasmid) along with the adjuvant CT (1 μg/μl). For the immunization, the animals were anesthetized with isoflurane and primed with final dose of 60 μg of DNA per mice followed by 2 and 4 weeks boosts using same dose without CT. In the case of the individual candidate immunizations, the animals received a total of 60 μg of individual plasmid. In CT control group, priming was with CT followed by TE boosts. One week after the last boost, blood and fecal samples were collected to monitor mucosal antibody response.

Challenging the Immunized Mice.

To determine the protective ability of the potential DNA vaccine candidates, all immunized mice were challenged with a dose of 5×10⁹ CFU of the streptomycin resistant EHEC O157:H7 strain 86-24, via gavage, two weeks after the last boost. Two hours prior to the challenge, mice received an i.p. dose of cimetidine hydroxyzine (10 mg/ml) to reduce stomach's acidity. Fecal samples were collected from each group at indicated days after infection. Stools were dissolved in 2 ml of PBS, serially diluted and plated. To recover bacteria from the intestine, the mice were euthanized at indicated days and ceca were excised and homogenized in 2 ml of PBS. Bacterial suspensions were serially-diluted and plated. Both organ and fecal samples were plated on MacConkey agar plates containing streptomycin and then incubated at 37° C. overnight prior to E. coli 0157:H7 colonies enumeration.

Sera and Feces Collection for Ig Determination.

Sera were obtained by retro-orbital bleeding, clotting whole blood for 30 min at room temperature and centrifugation at 3,000×g for 15 min at 4° C. The resulting supernatant was collected and used for ELISA. For sIgA measurement, feces were weighted and diluted to 1 g/ml with PBS-PM SF. After vigorous homogenization with vortex, feces were incubated for 1 h on ice and centrifuged at 4000 rpm for 30 min at 4° C. The supernatant was collected and stored at −20° C. prior to use.

Immune Response:

ELISA assay. Total serum IgG antibody responses were determined by ELISA according to manufacturer's instructions (ebiosciences). Briefly, polystyrene 96-well high binding ELISA plates (Nunc, Denmark) were coated overnight at 4° C. with capture IgG antibody. The plates were washed thrice in PBS containing 0.05% Tween 20 (v/v) (PBS-T) and plated with blocking buffer. The diluted serum sample (1:1000 and 1:10,000) and IgG standard of known activity were incubated, followed by repeated washing with 0.05% PBS-T. Next, horseradish peroxidase goat anti-mouse IgG in PBS-T (1:250) was added to the ELISA plates and incubated at 37° C. for 30 min followed by washing. One hundred μl of tetramethylbenzidine (TMB) were added to the cells and incubated at room temperature for 15 min. The reaction was stopped with 100 μl of 2 M H₂SO₄ and the OD₄₅₀ was determined.

For EHEC specific antibody response, wild-type EHEC O157:H7 were grown overnight in LB broth. Bacterial cells were pelleted (15 min at 5,000×g) and re-suspended in PBS. The bacterial suspension was pulse-sonicated on ice for 5 min. The sonicated sample was centrifuged (10,000×g for 15 min at 4° C.) and total protein concentration in the supernatant was determined by the bicinchoninic acid protein (BCA) assay. For ELISA, polystyrene 96 well Nunc plates were coated overnight at 4° C. with 100 μl of EHEC extract (2 μg/ml in coating buffer) and followed the procedure described above.

Bio-Plex Assay.

The cytokine levels in serum and feces from immunized and non-immunized mice were measured on a Bio-Plex 200 system powered by Luminex xMAP technology (Bio-Rad, USA) using a specific 23 panel group mouse assay kits (Cat. No. M60009RDPD), following manufacturer's instructions. The cytokines, chemokines and growth factors include: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p40), IL-12p70, IL-13, IL-17, TNF-α, IFN-γ, MIP-1α, MIP-1β, KC, eotaxin, MCP-1, G-CSF, GM-CSF and RANTES. A heatmap of a normalized matrix was created that correlates cytokine response pattern to stimulation by high priority vaccine candidates. For each cytokine, mean and standard deviation were calculated from their induction values (pg/ml) across the three candidates and were normalized to controls (TE and pVAX-1). The Z-score transformation was calculated for each cytokine by subtracting each induction value from the row mean and dividing by the row standard deviation (48). Overall, the Z-score gives an estimation of the deviation of the measurement from the row mean in standard deviation units. Each block of red or green represents a high positive or negative correlation between the cytokine production and the vaccine candidate under investigation.

Statistical Analysis.

All the statistical significance between control and vaccinated groups was assessed using SPSS software. One way ANOVA and Student's t-test with threshold of P<0.05 was used to analyze the data for colonization and antibody response. For the antibody responses the P value is indicated in the respective figures.

II. Vaccine Compositions

Vaccine compositions described herein may include adjuvants to further increase the immunogenicity of one or more of the EHEC antigens. Such adjuvants include any compound or compounds that act to increase an immune response to an EHEC antigen or combination of antigens, thus reducing the quantity of antigen necessary in the vaccine, and/or the frequency of injection necessary in order to generate an adequate immune response. Adjuvants may include for example, emulsifiers, muramyl dipeptides, avridine, aqueous adjuvants such as aluminum hydroxide, chitosan-based adjuvants, and any of the various saponins, oils, and other substances known in the art, such as Amphigen, LPS, bacterial cell wall extracts, bacterial DNA, synthetic oligonucleotides and combinations thereof (Schijns et al., Curr. Opi. Immunol. (2000) 12:456), Mycobacterial phlei (M. phlei) cell wall extract (MCWE) (U.S. Pat. No. 4,744,984), M. phlei DNA (M-DNA), M-DNA-M. phlei cell wall complex (MCC). For example, compounds which may serve as emulsifiers herein include natural and synthetic emulsifying agents, as well as anionic, cationic and nonionic compounds. Among the synthetic compounds, anionic emulsifying agents include, for example, the potassium, sodium and ammonium salts of lauric and oleic acid, the calcium, magnesium and aluminum salts of fatty acids (i.e., metallic soaps), and organic sulfonates such as sodium lauryl sulfate. Synthetic cationic agents include, for example, cetyltrimethylammonium bromide, while synthetic nonionic agents are exemplified by glyceryl esters (e.g., glyceryl monostearate), polyoxyethylene glycol esters and ethers, and the sorbitan fatty acid esters (e.g., sorbitan monopalmitate) and their polyoxyethylene derivatives (e.g., polyoxyethylene sorbitan monopalmitate). Natural emulsifying agents include acacia, gelatin, lecithin and cholesterol.

Other suitable adjuvants can be formed with an oil component, such as a single oil, a mixture of oils, a water-in-oil emulsion, or an oil-in-water emulsion. The oil may be a mineral oil, a vegetable oil, or an animal oil. Mineral oil, or oil-in-water emulsions in which the oil component is mineral oil are preferred. In this regard, a “mineral oil” is defined herein as a mixture of liquid hydrocarbons obtained from petrolatum via a distillation technique; the term is synonymous with “liquid paraffin,” “liquid petrolatum” and “white mineral oil.” The term is also intended to include “light mineral oil,” i.e., an oil which is similarly obtained by distillation of petrolatum, but which has a slightly lower specific gravity than white mineral oil. See, e.g., Remington's Pharmaceutical Sciences, supra. A particularly preferred oil component is the oil-in-water emulsion sold under the trade name of EMULSIGEN PLUS™ (comprising a light mineral oil as well as 0.05% formalin, and 30 mcg/mL gentamicin as preservatives), available from MVP Laboratories, Ralston, Nebr. Suitable animal oils include, for example, cod liver oil, halibut oil, menhaden oil, orange roughy oil and shark liver oil, all of which are available commercially. Suitable vegetable oils, include, without limitation, canola oil, almond oil, cottonseed oil, corn oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil, and the like.

Alternatively, a number of aliphatic nitrogenous bases can be used as adjuvants with the vaccine formulations. For example, known immunologic adjuvants include amines, quaternary ammonium compounds, guanidines, benzamidines and thiouroniums (Gall, D. (1966) Immunology 11:369-386). Specific compounds include dimethyldioctadecylammonium bromide (DDA) (available from Kodak) and N,N-dioctadecyl-N,N-bis(2-hydroxyethyl)propanediarnine (“avridine”). The use of DDA as an immunologic adjuvant has been described; see, e.g., the Kodak Laboratory Chemicals Bulletin 56(1):1-5 (1986); Adv. Drug Deliv. Rev. 5(3):163-187 (1990); J. Controlled Release 7:123-132 (1988); Clin. Exp. Immunol. 78(2):256-262 (1989); J. Immunol. Methods 97(2):159-164 (1987); Immunology 58(2):245-250 (1986); and Int. Arch. Allergy Appl. Immunol. 68(3):201-208 (1982). Avridine is also a well-known adjuvant. See, e.g., U.S. Pat. No. 4,310,550 to Wolff, III et al., which describes the use of N,N-higher alkyl-N′,N′-bis(2-hydroxyethyl)propane diamines in general, and avridine in particular, as vaccine adjuvants. U.S. Pat. No. 5,151,267 to Babiuk, and Babiuk et al. (1986) Virology 159:57-66, also relate to the use of avridine as a vaccine adjuvant.

The compositions of the present invention are normally prepared as injectables, either as liquid solutions or suspensions, or as solid forms which are suitable for solution or suspension in liquid vehicles prior to injection. The preparation may also be prepared in solid form, emulsified or the active ingredient encapsulated in liposome vehicles or other particulate carriers used for sustained delivery. For example, the vaccine may be in the form of an oil emulsion, water in oil emulsion, water-in-oil-in-water emulsion, site-specific emulsion, long-residence emulsion, sticky-emulsion, microemulsion, nanoemulsion, liposome, microparticle, microsphere, nanosphere, nanoparticle and various natural or synthetic polymers, such as nonresorbable impermeable polymers such as ethylenevinyl acetate copolymers and Hytrel® copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures, that allow for sustained release of the vaccine.

Furthermore, the polypeptides may be formulated into compositions in either neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 18th edition, 1990.

The composition is formulated to contain an effective amount of EHEC antigen(s) described herein, the exact amount being readily determined by one skilled in the art, wherein the amount depends on the animal to be treated and the capacity of the animal's immune system to synthesize antibodies. The composition or formulation to be administered will contain a quantity of one or more EHEC antigens adequate to achieve the desired state in the subject being treated. For purposes of the present invention, a therapeutically effective amount of a vaccine comprising EHEC antigens, contains about 0.05 to 1500 μg EHEC protein, preferably about 10 to 1000 μg EHEC protein, more preferably about 30 to 500 μg and most preferably about 40 to 300 μg, or any integer between these values.

Routes of administration include, but are not limited to, nasal, oral, topical, subcutaneous, intramuscular, intravenous, subcutaneous, intradermal, transdermal and subdermal. Depending on the route of administration, the volume per dose is preferably about 0.001 to 10 ml, more preferably about 0.01 to 5 ml, and most preferably about 0.1 to 3 ml. Vaccine can be administered in a single dose treatment or in multiple dose treatments (boosts) on a schedule and over a time period appropriate to the age, weight and condition of the subject, the particular vaccine formulation used, and the route of administration.

Any suitable pharmaceutical delivery means may be employed to deliver the compositions to the vertebrate subject. For example, inhalers, instillation devices, conventional needle syringes, spring or compressed gas (air) injectors (U.S. Pat. No. 1,605,763 to Smoot; U.S. Pat. No. 3,788,315 to Laurens; U.S. Pat. No. 3,853,125 to Clark et al.; U.S. Pat. No. 4,596,556 to Morrow et al.; and U.S. Pat. No. 5,062,830 to Dunlap), liquid jet injectors (U.S. Pat. No. 2,754,818 to Scherer; U.S. Pat. No. 3,330,276 to Gordon; and U.S. Pat. No. 4,518,385 to Lindmayer et al.), and particle injectors (U.S. Pat. No. 5,149,655 to McCabe et al. and U.S. Pat. No. 5,204,253 to Sanford et al.) are all appropriate for delivery of the compositions.

III. Proteinaceous Compositions

Proteinaceous compositions of the invention include polypeptides and polypeptide segments of proteins encoded by the nucleic acids corresponding to the GI numbers of table 3. In certain embodiments, antigens can be engineered to include polypeptide variants of the antigens corresponding to the entries in table 3. As used herein, a “protein” or “polypeptide” refers to a polymer of amino acid residues. In some embodiments, a wild-type version of a protein or polypeptide are employed, however, in many embodiments, all or part of an antigen or polypeptide is absent or altered.

A “modified protein” or “modified polypeptide” or “variant protein” or “variant polypeptide” refers to a protein or polypeptide whose chemical structure or amino acid sequence is altered with respect to the wild-type or a reference protein or polypeptide. In some embodiments, a modified protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). The modified activity or function may be reduced, diminished, eliminated, enhanced, improved, or altered in some other way with respect to that activity or function in a wild-type protein or polypeptide. It is contemplated that a modified protein or polypeptide may be altered with respect to one activity or function yet retain wild-type or unaltered activity or function in other respects.

It is contemplated that polypeptides may be modified by truncation, rendering them shorter than their corresponding unaltered form or by fusion or domain shuffling which may render the altered protein longer.

Amino acid sequence variants of the polypeptides of the present invention can be substitutional, insertional, or deletion variants. A mutation in a gene encoding a polypeptide may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more non-contiguous or contiguous amino acids (i.e., segment) of a polypeptide, as compared to a wild-type or unaltered polypeptide or other reference polypeptide.

Deletion variants lack one or more residues of the native, unaltered, or wild-type protein. Individual residues can be deleted, or all or part of a domain (such as a catalytic or binding domain) can be deleted. Insertional mutants typically involve the addition of material at a non-terminal point in the polypeptide; a specific type of insert is a chimeric polypeptide that includes homologous or similar portions of a related protein in place of the related portion of a target protein. This may include the insertion of an immunoreactive epitope or simply one or more residues. Terminal additions, typically called fusion proteins, may also be generated.

Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitutions may be non-conservative such that a function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.

The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids. Amino acids codons include: Alanine (Ala, A) GCA, GCC, GCG, or GCU; Cysteine (Cys, C) UGC or UGU; Aspartic acid (Asp, D) GAC or GAU; Glutamic acid (Glu, E) GAA or GAG; Phenylalanine (Phe, F) UUC or UUU; Glycine (GIy, G) GGA, GGC, GGG or GGU; Histidine (His, H) CAC or CAU; Isoleucine (Ile, I) AUA, AUC, or AUU; Lysine (Lys, K) AAA or AAG; Leucine (Leu, L) UUA, UUG, CUA, CUC, CUG, or CUU; Methionine (Met, M) AUG; Asparagine (Asn, N) AAC or AAU; Proline (Pro, P) CCA, CCC, CCG, or CCU; Glutamine (Gln, Q) CAA or CAG; Arginine (Arg, R) AGA, AGG, CGA, CGC, CGG, or CGU; Serine (Ser, S) AGC, AGU, UCA, UCC, UCG, or UCU; Threonine (Thr, T) ACA, ACC, ACG, or ACU; Valine (Val, V) GUA, GUC, GUG, or GUU; Tryptophan (Trp, W) UGG; and Tyrosine (Tyr, Y) UAC or UAU.

It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5′ or 3′ sequences, and yet still be essentially as set forth herein, including having a certain biological activity. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either the 5′ or 3′ portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.

The following is a discussion based upon changing of the amino acids of a protein described herein to create an equivalent, or even an improved, molecule. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on receptor molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and in its underlying polynucleotide sequence, and nevertheless produce a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the nucleic acid sequences of the antigens described herein without appreciable loss of biological utility or activity of interest. In certain aspects the nucleic acid can be divided into segments encoding fragments of a parent polypeptide that retain antigenicity.

In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring a biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (0.5); histidine *−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Examples of substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

IV. Nucleic Acid Molecules

Certain embodiments are directed to compositions and methods that include polynucleotides that are capable of expressing all or part of an antigenic protein or polypeptide described herein or discoverable through the described methods. The polynucleotides may encode a peptide or polypeptide containing all or part of a antigenic amino acid sequence.

As used herein, the term an isolated “RNA, DNA, or nucleic acid segment” refers to a RNA, DNA, or nucleic acid molecule that has been isolated from total genomic DNA or other contaminants. In certain embodiments the polynucleotide has been isolated free of other nucleic acids.

The term “complementary DNA” or “cDNA” refers to DNA prepared using RNA as a template. There may be times when the full or partial genomic sequence is preferred.

Similarly, a polynucleotide encoding a polypeptide refers to a nucleic acid segment including coding sequences and, in certain aspects, regulatory sequences, isolated substantially away from other naturally occurring genes or protein encoding sequences. In this respect, the term “gene” is used for simplicity to refer to a nucleic acid unit encoding a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization). As will be understood by those in the art, this functional term includes genomic sequences, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants.

The nucleic acid segments used in the present invention, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol.

It is contemplated that the nucleic acid constructs of the present invention may encode full-length polypeptide(s) from any source or encode a truncated or modified version of the polypeptide(s). A nucleic acid sequence may encode a full-length polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy. A tag or other heterologous polypeptide may be added to a polypeptide-encoding sequence. The term “heterologous” refers to a polypeptide, polynucleotide, or segment thereof that is not the same as the modified polypeptide, polynucleotide, or found associated with or encoded by the naturally occurring bacteria.

The nucleic acid segments used in the present invention encompass modified nucleic acids that encode modified polypeptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency. Functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by humans may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity.

In certain other embodiments, the invention concerns isolated nucleic acid segments and recombinant vectors that include within their sequence a contiguous nucleic acid sequence from that shown in sequences identified herein (and/or incorporated by reference).

In various embodiments, the polynucleotide may be altered or mutated. Alterations or mutations may include insertions, deletions, substitutions, rearrangement, inversions. Where employed, mutagenesis of a polynucleotide can be accomplished by a variety of standard, mutagenic procedures (Sambrook et al, 2001). Mutation is the process whereby changes occur in the function or structure of an organism or molecule. Mutation can involve modification of the nucleotide sequence of a single gene, blocks of genes or whole genomes. Changes in single genes may be the consequence of point mutations that involve the removal, addition, or substitution of a single nucleotide base within a DNA sequence, or they may be the consequence of changes involving the insertion or deletion of large numbers of nucleotides.

Insertional mutagenesis is based on the modification of a gene via insertion of a known nucleotide or nucleic acid fragment. Insertional mutagenesis may be accomplished using standard molecular biology techniques.

Structure-guided site-specific mutagenesis represents a powerful tool for the dissection and engineering of protein-ligand interactions (Wells, 1996; Braisted et al., 1996). The technique provides for the preparation and testing of sequence variants by introducing one or more nucleotide sequence changes into a selected DNA.

The term “expression vector” refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are translated into a protein, polypeptide, or peptide. Expression vectors can contain a variety of “control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra. In certain embodiments an expression vector is pVAX vector.

A “promoter” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements that bind regulatory proteins and molecules, such as RNA polymerase and other transcription factors. The phrases “operatively positioned,” “operatively coupled,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence. A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Heterologous translational control signals, including the ATG initiation codon, may need to be provided. The translational control signal and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.

Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites any of which can be used in conjunction with standard recombinant technology to digest the vector. (See Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997, incorporated herein by reference.) “Restriction enzyme digestion” refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. A vector can be linearized or fragmented using a restriction enzyme that cuts within the MCS to enable heterologous sequences to be ligated to the vector. “Ligation” refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other.

The vectors or constructs can comprise at least one termination signal. A “termination signal” or “terminator” is comprised of the nucleic acid sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels. Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.

A polyadenylation signal can be used to effect proper polyadenylation of a transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention. Embodiments include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.

In certain embodiments of the invention, cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker. Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable and screenable markers are well known to one of skill in the art.

V. Kits Related to EHEC Antigens

In still another embodiment, the present invention provides a pharmaceutical kit for ready administration of an immunogenic, prophylactic, or therapeutic regimen. This kit is designed for use in a method of inducing a high level of antigen-specific immune response in a mammalian or vertebrate subject. The kit may contain at least one immunogenic composition comprising an antigenic composition as described herein. For example, multiple prepackaged dosages of the immunogenic composition or a DNA vector encoding antigens are provided in the kit for multiple administrations. The kit may also contain at least one immunogenic composition comprising an EHEC immunogenic composition as described herein. In one embodiment, multiple prepackaged dosages of the expression vectors and/or immunogenic composition are provided in the kit for multiple administrations.

The kit also contains instructions for using the immunogenic compositions in a prime/boost method as described herein. The kits may also include instructions for performing certain assays, various carriers, excipients, diluents, adjuvants and the like above-described, as well as apparatus for administration of the compositions, such as syringes, electroporation devices, spray devices, etc. Other components may include disposable gloves, decontamination instructions, applicator sticks or containers, among other compositions.

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1. An immunogenic composition comprising a polypeptide having an amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 in a therapeutic formulation.
 2. The composition of claim 1 comprising a polypeptide having an amino acid sequence of SEQ ID NO:1 and a polypeptide having an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3.
 3. The composition of claim 1 comprising a polypeptide having an amino acid sequence of SEQ ID NO:2 and a polypeptide having an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3.
 4. The composition of claim 1 comprising a polypeptide having an amino acid sequence of SEQ ID NO:3 and a polypeptide having an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
 5. The composition of claim 1 comprising a polypeptide having an amino acid sequence of SEQ ID NO:1, a polypeptide having an amino acid sequence of SEQ ID NO:2, and a polypeptide having the amino acid sequence of SEQ ID NO:3.
 6. A method for stimulating an immune response to an Enterohemorrhagic Escherichia coli (EHEC) in a subject comprising providing an immunogenic composition of claim
 1. 7. The method of claim 6, wherein the polypeptides are encoded by an expression vector.
 8. The method of claim 6, wherein the subject is bovine.
 9. The method of claim 6, wherein the subject is human. 